Novel human hepatoma associated protein and the polynucleotide encoding said polypeptide

ABSTRACT

The invention disclosed a new kind of human protein that associated with hepatoma and the polynucleotide encoding said polypeptide and a process for producing the polypeptide by recombinant methods. It also disclosed the method of applying the polypeptide for the diagnosis and treatment of various kinds of disease, such as cancer. The antagonist of the polypeptide and therapeutic use of the same is also disclosed. In addition, the invention disclosed the use of the same. In addition, the invention refers to the use of polynucleotide encoding said hepatoma associated human protein.

FIELD OF INVENTION

[0001] This invention relates to the field of biotechnology, and, inparticular, relates to the a novel polynucleotide encoding humanhepatoma associated protein and the polypeptide encoded by saidpolynucleotide. The invention also relates to the uses and preparationof these polynucleotides and polypeptides. The hepatoma associatedprotein of the invention is a suppresser of hepatocellular carcinoma.

PRIOR ART

[0002] The mortality rate of malignant tumor is just lower than that ofcardio- and cerebro-vascular disease. Hepatocarcinoma is one of the mostcommon tumors in China. The development of hepatocarcinoma is a complexprocess involving multiple genes and steps and effected by theactivation of many oncogenes and inactivation of anti-oncogenes. Theanti-oncogenes are much important. Therefore, to find anti-oncogenes areone of the focuses of the current studies. A high frequency of loss ofheterozygosity (LOH) on a segment of chromosome in a certain cancersuggests that there is an anti-oncogene associated with said cancer inthis segment.

[0003] There is a high frequency of loss of heterozygosity on chromosome17p13.3 in several tumors. Therefore, several laboratories in the worldhave tried to find out the anti-oncogenes in chromosome 17p13.3. Up tonow, three possible anti-oncogene (Hic-I, OVCA1 and OVCA2) have beenfound. Hic-I is highly methylated and lowly expressed in tumor tissues.OVCA1 and OVCA2 are lowly expressed or not expressed at all in oophoromatissue or cell lines. These three genes are all located near site YNZ22of the chromosome.

[0004] Since cancer is one of the main diseases harmful to human health,people are concerned about the early diagnosis and gene therapy ofcancer so as to effectively cure and prevent tumors, such ashepatocarcinoma. Therefore, there is a keen need in the art to developnew tumor-associated and/or tumor-inhibiting human proteins and theiragonist/antagonist.

SUMMARY OF INVENTION

[0005] One purpose of the invention is to provide a novel hepatomaassociated protein, which is named c63R protein, and its fragments,analogs and derivatives.

[0006] Another purpose of the invention is to provide a polynucleotideencoding said polypeptides.

[0007] Still another purpose of the invention is to provide a method forpreparing said polypeptides and the uses of said polypeptides and theirencoding sequences.

[0008] In the first aspect, the invention provides an isolated humanc63R polypeptide, which comprises a polypeptide having the amino acidsequence of SEQ ID NO: 2, its conservative variants, its activefragments, and its active derivatives. Preferably, said polypeptide is apolypeptide having the amino acid sequence of SEQ ID NO: 2.

[0009] In the second aspect, the invention provides an isolatedpolynucleotide, which comprises a nucleotide sequence sharing at least85% homology to the following nucleotide sequence: (a) the nucleotidesequence encoding the above c63R polypeptide; (b) the polynucleotidecomplementary to nucleotide sequence of (a). Preferably, said nucleotidesequence encodes a polypeptide comprising the amino acid sequence of SEQID NO: 2. More preferably, said polynucleotide comprises the ORFsequence or full-length sequence of SEQ ID NO: 1.

[0010] In the third aspect, the invention provides a vector comprisingthe above polynucleotide and a host cell transformed with said vector, ahost cell transformed with said polynucleotide.

[0011] In the fourth aspect, the invention provides a method forproducing a polypeptide having the activity of c63R protein, whichcomprises:

[0012] (a) culturing the above transformed host cell under theconditions suitable for the expression of protein;

[0013] (b) isolating the polypeptides having the activity of c63Rprotein from the culture.

[0014] In the fifth aspect, the invention provides an antibodyspecifically bound to c63R protein. Also provided are nucleic acidmolecules comprising consecutive 10-800 nucleotides of the abovepolynucleotide.

[0015] In the sixth aspect, the invention provides pharmaceuticalcomposition comprising a safe and efficient amount of hepatocarcinomaassociated c63R protein and pharmaceutically acceptable carrier. Saidpharmaceutical composition can be used for the therapy of diseases, suchas cancer and cell abnormal proliferation.

[0016] In the seventh aspect, the invention provides a method fordetecting the carcinomatous change or cancer susceptibility ofhepatocytes, comprising the steps of: detecting whether there is anychange of c63R transcript in the hepatocyte sample when compared withthe normal c63R transcript, and said change indicating that thehepatocyte has developed cancer or is cancer susceptible; or detectingwhether there is any change of activity of c63R protein in thehepatocyte sample when compared with the normal c63R protein, and saidchange indicating that the hepatocyte has developed cancer or is cancersusceptible. Preferably, said change is nucleotide deletion, insertionor substitution. More preferably, said change is selected from the groupconsisting of: deletion of nucleotides 395-481 in SEQ ID NO: 1, deletionof nucleotides 328-1511 in SEQ ID NO: 1, deletion of nucleotides1143-1231 in SEQ ID NO: 1, deletion of nucleotides 1325-1419 in SEQ IDNO: 1, change from C to T of nucleotide 1106 in SEQ ID NO: 1, deletionof nucleotide 1048 in SEQ ID NO: 1.

[0017] In the eighth aspect, the invention provides a kit for detectinghepatocarcinoma comprising: (1) a pair of primers specificallyamplifying human c63R gene, (2) the agents for detecting whether thereis any change between the amplification product and the normal c63Rgene.

[0018] The other aspects of the invention will be apparent to theskilled in the art in light of the technical disclosure of theinvention.

[0019]FIG. 1 shows the contig of genomic clones covering HCCS1 and thegene structure of HCCS1.

[0020] Markers used in genomic clone screening and contig constructionare shown at the top.

[0021] The contig of genomic clones covering HCCS1 is shown in themiddle. The exons of HCCS1 are shown at the bottom.

[0022]FIG. 2 shows the locations of HCCS1 in cells. FIG. 2A is afluorescence photo of NIH/3T3 cells transfected with pEGFP-HCCS1. FIG.2B is an immunofluorescence photo stained with anti-mitochondriaantibody of cells shown in FIG. 2a. FIG. 2c is the overlay image ofFIGS. 2a and 2 b, indicating the localization of HCCS1 is atmitochondria.

[0023]FIG. 3 shows the immunohistochemical staining of human HCC andsurrounding noncancerous tissues. HCC: hepatocarcinoma cell. HC,hepatocytes.

[0024]FIG. 4 shows the effect of HCCS1 expression on cell growth. FIG.4A, colony formation of SMMC-7721 cells transfected with vectorpcDNA3.1/V5-His. FIG. 4B, colony formation of SMMC-7721 cellstransfected with vector containing HCCS1 cDNA. FIG. 4C, diagram ofcolony formation assay in triplicate. P<0.01 in comparison with thevector group.

[0025]FIG. 5 shows the effect of HCCS1 on the tumor formation in nudemice. P<0.01 in comparison with GFP vector control group.

DETAILED DESCRIPTION OF INVENTION

[0026] In the hepatocarcinoma study, the inventors have found that theregion of LOH in chromosome 17p13.3 is located from YNZ22 site to D17S34of telomere. The minimum region of LOH is within the region betweenD17S643 and D17S1574. The inventors have cloned the genes in this regionand obtained c63R gene in D17S849. It has been found there are deletionand frameshift mutation of c63R gene in HCC tissue, confirming c63R geneis a HCC associated gene. Further experiments demonstrated that c63Rgene is a hepatocellular carcinoma suppresser so that c63R is alsoreferred as HCCS1 (Hepatocellular Carcinoma Suppresser 1). It has beenindicated in the studies that HCCS1 is located in mitochondria.Moreover, HCCS1 is negatively expressed in hepatocarcinoma cells andpositively expressed in normal hepatocyte. After transformed into HCCcell and nude mice, HCCS1 inhibits the growth of tumor cell andformation of tumor.

[0027] As used herein, the terms “c63R protein”, “c63R polypeptide”,“HCC associated protein c63R”, “HCCS1 protein”, “HCCS1 polypeptide”, and“Hepatocellular Carcinoma suppresser HCCS1” are changeable. Each of themmeans a protein or polypeptide having the amino acid sequence of humanHCC associated protein c63R (SEQ ID NO:2). These terms also include theHCC associated protein c63R having or not having the starting Metresidue.

[0028] As used herein, the term “isolated” refers to a substance whichhas been isolated from the original environment. For naturally occurringsubstance, the original environment is the natural environment. Forexample, the polynucleotide and polypeptide in a naturally occurringstate in the viable cells are not isolated or purified. However, if thesame polynucleotide and polypeptide have been isolated from othercomponents naturally accompanying them, they are isolated or purified.

[0029] As used herein, the terms “isolated HCC associated c63R proteinor polypeptide” or “isolated c63R protein or polypeptide” mean thathuman c63R protein does not essentially contain other proteins, lipids,carbohydrate or any other substances associated therewith in nature. Theskilled in the art can purify human c63R protein by standard proteinpurification techniques. Essentially purified polypeptide forms a singlemain band on a non-reductive PAGE gel. The purity of human c63R proteinpolypeptide can be analyzed by amino acid sequence analysis.

[0030] The polypeptide of invention may be a recombinant polypeptide,natural polypeptide, or synthetic polypeptide, preferably a recombinantpolypeptide. The polypeptide of invention may be a purified naturalproduct or a chemically synthetic product. Alternatively, it may beproduced from prokaryotic or eukaryotic hosts, such as bacteria, yeast,higher plant, insect, and mammal cells, using recombinant techniques.According to the host used in the protocol of recombinant production,the polypeptide of invention may be glycosylated or non-glycosylated.The polypeptide of invention may or may not comprise the starting Metresidue.

[0031] The invention further comprises the fragments, derivatives andanalogues of human c63R protein. As used in the invention, the terms“fragment”, “derivative” and “analogue” mean the polypeptide thatessentially retains the same biological functions or activity of HCCassociated human c63R protein of the invention. The fragment, derivativeor analogue of the polypeptide of invention may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues include a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretarysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0032] The polynucleotide according to the invention may be in the formsof DNA and RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, etc.,in single strand or double strand form. A single strand DNA may be anencoding strand or non-encoding strand. The coding sequence for maturepolypeptide may be identical to the coding sequence shown in SEQ ID NO:1, or is a degenerate sequence. As used herein, the term “degeneratesequence” means an sequence which encodes a protein or peptidecomprising a sequence of SEQ ID NO: 2 and which has a nucleotidesequence different from the sequence of coding region in SEQ ID NO: 1.

[0033] The sequences encoding the mature polypeptide include thoseencoding only the mature polypeptide, those encoding mature polypeptideplus various additional encoding sequence, the encoding sequence formature polypeptide plus the non-encoding sequence and optionaladditional encoding sequence.

[0034] The term “polynucleotide encoding the polypeptide” includes thepolynucleotide encoding said polypeptide and the polynucleotidecomprising additional and/or non-encoding sequence.

[0035] The invention further relates to the variants of the hereinabovepolynucleotides which encode a polypeptide having the same amino acidsequence of invention, or its fragment, analogue and derivative. Thevariant of the polynucleotide may be a naturally occurring allelicvariant of the polynucleotide or a non-naturally occurring variant ofthe polynucleotide. Such nucleotide variants include substitution,deletion, and insertion variants. As known in the art, the allelicvariant is a substitution form of polynucleotide, which may be asubstitution, deletion, and insertion of one or more nucleotides withoutsubstantially changing the functions of the encoded polypeptide.

[0036] The present invention further relates to polynucleotides, whichhybridize to the hereinabove-described sequences, if there is at least50% and preferably at least 70% identity between the sequences. Thepresent invention particularly relates to polynucleotides, whichhybridize under stringent conditions to the polynucleotides of theinvention. As herein used, the term “stringent conditions” means thefollowing conditions: (1) hybridization and washing under low ionicstrength and high temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; (2)hybridization after adding denaturants, such as 50% (v/v) formamide,0.1% bovine serum/0.1% Ficoll, 42° C.; or (3) hybridization of twosequences sharing at least 95%, preferably 97% homology. Further, thepolynucleotides which hybridize to the hereinabove describedpolynucleotides encode a polypeptide which retains the same biologicalfunction or activity as the mature polypeptide as set forth in SEQ IDNO: 2

[0037] The invention also relates to nucleic acid fragments hybridizedwith the hereinabove sequence. As used in the present invention, thelength of the “nucleic acid fragment” is at least 15 bp, preferably atleast 30 bp, more preferably at least 50 bp, and most preferably atleast 100 bp. The nucleic acid fragment can be used in the amplificationtechniques of nucleic acid, e.g., PCR, so as to determine and/or isolatethe polynucleotide encoding c63R protein.

[0038] The polypeptide and polynucleotide of the invention arepreferably in isolated form, preferably purified to be homogenous.

[0039] According to the invention, the DNA sequence encoding can beobtained in various ways. For example, the polynucleotide is isolated bythe hybridization techniques well-known in the art, which includes, butare not limited to 1) the hybridization between the probe and genomic orcDNA library so as to select the homologous polynucleotide sequence, and2) expression of the antibodies against the library so as to screen outthe DNA fragments having the common structure features.

[0040] The specific DNA fragment sequences encoding c63R protein mayfurther be obtained by the following methods so as to obtain thedouble-stranded DNA for said polypeptide: 1) isolating double-strandedDNA sequence from genomic DNA; and 2) chemical synthesis of DNAsequence.

[0041] In the above methods, the isolation of genomic DNA is leastfrequently used. The direct chemical synthesis of DNA sequence iscommonly used when the whole amino acid sequence of the desiredpolypeptide product is known. When the whole amino acid sequence of thedesired polypeptide product is not known, the direct chemical synthesisof DNA sequence is impossible and the method is to isolate cDNAsequence. The standard method for isolating the cDNA of interest is toisolate mRNA from donor cells that highly express said gene followed byreverse transcription of mRNA to form plasmid or phage cDNA library.There are many sophisticated techniques for extracting mRNA and the kitsare commercially available (Qiagene). The conventional method can beused to construct cDNA library (Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). ThecDNA libraries are also commercially available, e.g., the different cDNAlibrary from Clontech. When PCR is used in combination, even anextremely small amount of expression products can be cloned.

[0042] The conventional methods can be used for screening the gene ofinvention from a library. These methods include, but are not limited to,(1) DNA-DNA or DNA-RNA hybridization, (2) the appearance or loss of thefunction of marker gene, (3) the determination of the level of c63Rtranscripts, (4) the determination of protein product of gene byimmunology methods or the biological activity assays. These methods canbe used alone or in combination.

[0043] In method (1), the probe used in the hybridization could beidentical to any portion of polynucleotide of invention. The length ofprobe is typically at least 15, preferably at least 30, more preferablyat least 50, and most preferably at least about 100 nucleotides.Usually, the length of probe is less than 2 kb, preferably 1 kb. Theprobe usually is the DNA sequence chemically synthesized on the basis ofthe sequence information of gene of invention. Of course, the gene ofinvention itself or the fragment thereof can be used as a probe. Thelabels for DNA probe include, e.g., radioactive isotopes, fluoresceinsor enzymes, such as alkaline phosphatase.

[0044] In method (4), the detection of the protein products expressed byc63R gene can be carried out by immunology methods, such as Westernblotting, radioimmunoassay, and ELISA.

[0045] The method of amplification of DNA/RNA by PCR (Saiki, et al.Science 1985; 230:1350-1354) is preferably used to obtain the gene ofthe invention. Especially when it is difficult to obtain the full-lengthcDNA, the method of RACE is preferably used. The primers used in PCR canbe properly selected according to the polynucleotide sequenceinformation of invention disclosed herein and synthesized by theconventional methods. The amplified DNA/RNA fragments can be isolatedand purified by conventional methods such as gel electrophoresis.

[0046] For the gene of the invention or its DNA fragments, thesequencing of polynucleotide sequence can be carried out by theconventional dideoxy sequencing method (Sanger et al. PNAS, 1977, 74:5463-5467). The sequencing of polynucleotide sequence can also becarried out using the commercially available sequencing kits. In orderto obtain the full-length cDNA sequence, it is necessary to repeat thesequencing. Sometimes, it may sequence the DNA of several clones toobtain the full-length cDNA sequence.

[0047] The invention further relates to a vector comprising thepolynucleotide of the invention, a genetic engineered host celltransformed with the vector of the invention or directly with thesequence encoding c63R protein, and the method for producing thepolypeptide of invention by recombinant techniques.

[0048] The recombinant c63R polypeptides can be expressed or produced bythe conventional recombinant DNA technology (Science, 1984; 224:1431),using the polynucleotide sequence of invention. Generally, it comprisesthe following steps:

[0049] (1) transfecting or transforming the appropriate host cells withthe polynucleotide encoding c63R polypeptide of the invention or thevector containing said polynucleotide;

[0050] (2) culturing the host cells in an appropriate medium;

[0051] (3) isolating or purifying the protein from the medium or cells.

[0052] In the present invention, the polynucleotide sequences encodinghuman c63R protein may be inserted into a recombinant expression vector.The term “expression vector” refers to a bacterial plasmid,bacteriophage, yeast plasmid, plant virus or mammal cell virus, such asadenovirus, retrovirus or any other vehicle known in the art. Vectorssuitable for use in the present invention include, but are not limitedto, the T7-based expression vector for expression in bacteria(Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vectorfor expression in mammalian cells (Lee and Nathans, J Biol. Chem.,263:3521, 1988) and baculovirus-derived vectors for expression in insectcells. On the whole, any plasmid or vector can be used to construct therecombinant expression vector as long as it can replicate and is stablein the host. One important feature of expression vector is that theexpression vector typically contains an origin of replication, apromoter, a marker gene as well as the translation regulatorycomponents.

[0053] The methods known by the artisans in the art can be used toconstruct an expression vector containing the DNA sequence of c63R andappropriate transcription/translation regulatory components. Thesemethods include in vitro recombinant DNA technique, DNA synthesistechnique, in vivo recombinant technique and so on (Sambroook, et al.Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989). The DNA sequence is efficiently linked to the properpromoter in an expression vector to direct the synthesis of mRNA. Theexemplary promoters are lac or trp promoter of E.coli; P_(L) promoter ofλ phage; eukaryotic promoter including CMV immediate early promoter, HSVthymidine kinase promoter, early and late SV40 promoter, LTRs ofretrovirus and some other known promoters which control the geneexpression in the prokaryotic cells, eukaryotic cells or virus. Theexpression vector may further comprise a ribosome binding site forinitiating the translation, transcription terminator and the like.

[0054] Further, the expression vector preferably comprises one or moreselective marker genes to provide a phenotype for the selection of thetransformed host cells, e.g., the dehydrofolate reductase, neomycinresistance gene and GFP (green flurencent protein) for eukaryotic cells,as well as tetracycline or ampicillin resistance gene for E. coli.

[0055] The vector containing said DNA sequence and proper promoter orregulatory elements can be transformed into appropriate host cells toexpress the protein.

[0056] The “host cell” includes prokaryote, such as bacteria; primaryeukaryote, such as yeast; advanced eukaryotic, such as mammalian cells.The representative examples are bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; plantcells; insect cells such as Drosophila S2 or Sf9; animal cells such asCHO, COS or Bowes melanoma, etc.

[0057] Transcription of the polynucleotide of invention in highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase the gene transcription.Examples include the SV40 enhancer on the late side of the replicationorigin 100 to 270 bp, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

[0058] The skilled in the art know clearly how to select appropriatevectors, promoters, enhancers and host cells.

[0059] Recombinant transformation of host cell with the DNA sequence ofinvention might be carried out by conventional techniques well known tothose skilled in the art. Where the host is prokaryotic such as E. coli,the competent cells, which are capable of DNA uptake, can be preparedfrom cells harvested after exponential growth phase and subsequentlytreated by the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ can be used. The transformation can also be carriedout by electroporation, if desired. When the host is an eukaryote, suchmethods of transfection of DNA as calcium phosphate co-precipitates,conventional mechanical procedures such as micro-injection,electroporation, or liposome-mediated transfection may be used.

[0060] The transformants are cultured using conventional methods toexpress the polypeptides of the invention. According to the used hostcells, the medium for cultivation can be selected from variousconventional mediums. The host cells are cultured under a conditionsuitable for its growth until the host cells grow to an appropriate celldensity. Then, the selected promoter is induced by appropriate means(e.g., temperature shift or chemical induction) and cells are culturedfor an additional period.

[0061] In the above methods, the recombinant polypeptide may be includedin the cells, or expressed on the cell membrane, or secreted out of thecell. If desired, the physical, chemical and other properties can beutilized in various isolation methods to isolate and purify therecombinant protein. These methods are well-known to those skilled inthe art and include, but are not limited to conventional renaturationtreatment, treatment by protein precipitant (such as saltprecipitation), centrifugation, cell lysis by osmosis, sonication,supercentrifugation, molecular sieve chromatography or gelchromatography, adsorption chromatography, ion exchange chromatagraphy,HPLC, and any other liquid chromatagraphy, and the combination thereof.

[0062] The studies of the invention showed that, in the canerous cells,almost all of c63R genes existed mutations including deletions,insertions and substitution in the c63R encoding sequence and non-codingsequence. These mutations make c63R protein have no or low activity inhepatocyte, finally directly or indirectly causing HCC.

[0063] Therefore, the recombinant HCC associated c63R protein orpolypeptide have various uses including, but not to be limited to:curing disorders (e.g., HCC) caused by low or no activity of c63Rprotein, and screening out antibodies, polypeptides or ligands asagonists or antagonists of c63R. For example, antibodies can be used toactivate or inhibit the function of c63R protein. The expressedrecombinant c63R protein can be used to screen polypeptide library tofind out therapeutically valuable polypeptide molecules which inhibit oractivate c63R protein.

[0064] The invention also provides the method for screening compounds soas to identify agents which improve c63R protein (agonists) or repressc63R protein (antagonists). For example, in the presence of an agent,the mammal cells or the membrane preparation expressing c63R protein canbe incubated with the labeled c63R protein to determine the ability ofthe agent to enhance or repress the interaction.

[0065] The antagonists of c63R protein include the screened antibodies,compounds, receptor deletants and analogues. The antagonists of c63Rprotein can bind to c63R protein and eliminate its function, or inhibitthe production of c63R, or bind to the active site of said polypeptideso that the polypeptide can not function biologically.

[0066] When screening the compound as an antagonist, c63R protein may beadded into the biological assay. One can determine whether the compoundis an antagonist by determining the its effect on the interactionbetween c63R protein and its receptor. Using the method same as that forscreening compounds, one can screen out the receptor deletants andanalogues acting as antagonists.

[0067] The polypeptide of invention can be directly used for thetreatment of diseases, e.g., various malignant tumors, abnormal cellproliferation, especially HCC.

[0068] The polypeptide, and its fragment, derivative, analogue or cellscan be used as antigens to produce antibodies. These antibodies may bepolyclonal or monoclonal antibodies. The polyclonal antibodies can beprepared by immunizing animals with c63R protein. The techniques forproducing monoclonal antibodies against c63R protein include, but arenot limited to, the hybridoma technique, the trioma technique, the humanB-cell hybridoma technique, the EBV-hybridoma technique and so on.

[0069] According to the invention, the polypeptides, and its antagonistsmay be employed in combination with a suitable pharmaceutical carrier.Such a carrier includes but is not limited to water, glucose, ethanol,salt, buffer, glycerol, and combinations thereof. Such compositionscomprise a safe and effective amount of the polypeptide or antagonist,as well as a pharmaceutically acceptable carrier or excipient which doesnot influence the effect of the drug. These compositions can be used fortreatment of disease.

[0070] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container (s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In addition, the polypeptides of the invention may beemployed in conjunction with other therapeutic compounds.

[0071] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. The c63Rprotein is administered in an amount, which is effective for treatingand/or prophylaxis of the specific indication. The amount of c63Rprotein administrated on patient will depend upon various factors, suchas delivery methods, the subject health, and the like, and is within thejudgment of the skilled clinician.

[0072] c63R polynucleotides also have many therapeutic applications.Gene therapy technology can be used in the therapy of the abnormal cellproliferation, development or metabolism, which is caused by the loss ofc63R expression or the expression of abnormal or non-active c63R.Recombinant gene therapy vectors, such as virus vectors, can be designedto express mutated c63R so as to inhibit the activity of endogenousc63R. One form of the mutated c63R is a truncated c63R whose signaltransduction domain is deleted. Therefore, this mutated c63R can bindthe downstream substrate without the activity of signal transduction.Thus, the recombinant gene therapy vectors can be used to cure thediseases caused by the abnormal expression or activity of c63R. Theexpression vectors derived from virus, such as retrovirus, adenovirus,adeno-associated virus, herpes simplex virus, parvovirus, and so on, canbe used to introduce the c63R gene into the cells. The methods forconstructing a recombinant virus vector harboring c63R gene aredescribed in the literature (Sambrook, et al.). In addition, therecombinant c63R gene can be packed into liposome and then transferredinto the cells.

[0073] Also included in the invention are ribozyme and theoligonucleotides, including antisense RNA and DNA, which inhibit thetranslation of c63R mRNA. Ribozyme is an enzyme-like molecule capable ofspecifically cutting certain RNA. The mechanism is the nucleic acidendo-cleavage after the specific hybridization of ribozyme molecule andthe complementary target RNA. Antisense RNA and DNA as well as ribozymecan be prepared by using any conventional techniques for RNA and DNAsynthesis, e.g., the widely used solid phase phosphite chemical methodfor oligonucleotide synthesis. Antisense RNA molecule can be obtained bythe in vivo or in vitro transcription of the DNA sequence encoding saidRNA, wherein said DNA sequence is integrated into the vector and in thedownstream of RNA polymerase promoter. In order to increase stability,the nucleic acid molecules can be modified in many manners, e.g.,increasing the length of the flanking sequences, replacing thephosphodiester bond with the phosphothioester bond in theoligonucleotide.

[0074] The methods for introducing the polynucleotides into tissues orcells include: directly injecting the polynucleotides into tissue in thebody, in vitro introducing the polynucleotides into cells with vectors,such as virus, phage, or plasmid, and then transplanting the cells intothe body.

[0075] The polypeptide of invention is useful in the analysis of peptidespectrum. For example, the polypeptide can be specifically cut byphysical, chemical, or enzymatic means, and then analyzed by one, two orthree dimensional gel electrophoresis.

[0076] The invention also provides the antibodies against thedeterminants of c63R protein. These antibodies include, but are notlimited to, polyclonal antibody, monoclonal antibody, chimeric antibody,single-chain antibody, Fab fragment and the fragments produced by Fabexpression library.

[0077] The antibody against c63R protein can be used inimmunohistochemical method to detect the presence of c63R protein in thebiopsy specimen.

[0078] The monoclonal antibody specific to c63R protein can be labeledby radioactive isotopes, and injected into human body to trace thelocation and distribution of c63R protein. This radioactively labeledantibody can be used in the non-wounding diagnostic method for themapping of the tumor and determination of the metastasis of tumor cells.

[0079] The antibody of the invention is useful for the therapy or theprophylaxis of disorders related to the c63R protein. The appropriateamount of antibody can be administrated to stimulate or block theproduction or activity of c63R protein.

[0080] Antibodies can also be designed as an immunotoxin targeting atthe particular site in the body. For example, a monoclonal antibodyhaving high affinity to c63R protein can be covalently bound tobacterial or plant toxins, such as diphtheria toxin, ricin, ormosine.One common method is to challenge the amino group on the antibody withsulfydryl cross-linking agents, such as SPDP, and bind the toxin ontothe antibody by interchanging the disulfide bonds. This hybrid antibodycan be used to kill c63R protein-positive cells.

[0081] The polyclonal antibodies can be prepared by immunizing animals,such as rabbit, mouse, and rat, with c63R protein. Various adjuvants,e.g., Freund's adjuvant, can be used to enhance the immunization.

[0082] The techniques for producing c63R protein monoclonal antibodiesinclude the hybridoma technique (Kohler and Milstein. Nature, 1975,256:495-497). The chimeric antibody comprising a constant region ofhuman origin and a variable region of non-human origin can be producedusing the conventional method in the art (Morrison et al, PNAS,1985,81:6851). Furthermore, the techniques for producing single-chainantibody (U.S. Pat. No. 4,946,778) are also useful for preparing thesingle-chain antibody against c63R protein.

[0083] The polypeptide molecule capable of binding to c63R protein canbe obtained by screening out the random polypeptide library consistingof the various combinations of amino acids bound onto the solid matrix.Typically, c63R protein is labeled in the screening.

[0084] The invention further provides diagnostic assays for quantitativeand in situ measurement of c63R protein level. These assays are wellknown in the art and include FISH assay and radioimmunoassay. The levelof c63R protein detected in the assay can be used to illustrate theimportance of c63R protein in diseases and to determine the diseasesassociated with c63R protein.

[0085] The polynucleotide encoding c63R protein can be used in thediagnosis of c63R protein related diseases. The polynucleotide encodingc63R can be used to detect whether c63R is expressed or not, and whetherthe expression of c63R is normal or abnormal, e.g., in the case ofdiseases. c63R DNA sequences can be used in the hybridization withbiopsy samples to determine the expression of c63R. The hybridizationmethods include Southern blotting, Northern blotting and in situblotting, etc., which are public and sophisticated techniques. Thecorresponding kits are commercially available. A part of or all of thepolynucleotides of the invention can be used as probe and fixed on amicroarray or DNA chip for analysis the differential expression of genesin tissues and for the diagnosis of genes. The c63R specific primers canbe used in RNA-polymerase chain reaction and in vitro amplification todetect the transcripts of c63R.

[0086] Further, detection of the mutation of c63R gene is useful for thediagnosis of c63R protein related diseases. The mutation forms of c63Rinclude site mutation, translocation, deletion, rearrangement and anyother mutations compared with the wild-type c63R DNA sequence (e.g., thenormal sequence of SEQ ID NO: 1). The conventional methods, such asSouthern blotting, DNA sequencing, PCR and in situ blotting, can be usedto detect mutation. Moreover, mutation sometimes affects the expressionof protein. Therefore, Northern blotting and Western blotting can beused to indirectly determine whether the gene is mutated or not.

[0087] The full length c63R nucleotide sequence or its fragment of theinvention can be prepared by PCR amplification, recombinant method andsynthetic method. For PCR amplification, one can obtain said sequencesby designing primers based on the nucleotide sequence disclosed in theinvention, especially the sequence of ORF, and using cDNA librarycommercially available or prepared by routine techniques known in theart as a template. When the sequence is long, it is usually necessary toperform two or more PCR amplifications and link the amplified fragmentstogether in the correct order.

[0088] Once the sequence is obtained, a great amount of the sequencescan be produced by recombinant methods. Usually, said sequence is clonedin a vector which is then transformed into a host cell. Then thesequence is isolated from the amplified host cells using conventionaltechniques.

[0089] Further, the sequence can be produced by synthesis, especiallywhen the fragment is short. Typically, several small fragments aresynthesized and linked together to obtain a long sequence.

[0090] At present, it is completely feasible to chemically synthesizethe DNA sequence encoding the protein of the invention, or the fragmentsor derivatives thereof. In addition, the mutation can be introduced intothe sequence of the protein by chemical synthesis.

[0091] Since the c63R protein of invention has the nature amino acidsequence from human, it is predicted that, compared with the otherproteins in the same family from other animals, it has higher activityand/or lower side-effect when administrated to human. For example, thereis no or low immunogenicity in vivo.

[0092] The invention is further illustrated by the following examples.It is appreciated that these examples are only intended to illustratethe invention, but not to limit the scope of the invention. For theexperimental methods in the following examples, they are performed underroutine conditions, e.g., those described by Sambrook. et al., inMolecule Clone: A Laboratory Manual, New York: Cold Spring HarborLaboratory Press, 1989, or as instructed by the manufacturers, unlessotherwise specified.

EXAMPLE 1 Determination of LOH Region in Chromosome 17p13.3 in HCC

[0093] In this Example, the analysis of LOH in HCC tissue was carriedout by using polymorphic markers in chromosome 17p13.3, so as todetermine the LOH region and the minimum frequent LOH region.

[0094] 1. HCC and Noncancerous Tissue Samples:

[0095] The HCC and noncancerous tissues of 54 primary HCC cases wereobtained from Qi Dong Liver Cancer Institute. The tissues were cut inthe operation, immediately frozen, and stored at −80° C. inrefrigerator.

[0096] 2. Extraction of Tissue DNA

[0097] The tissues were taken out from the low-temperature refrigerator,placed in a mortar-grinder and milled. The extraction of tissue DNA werecarried out according to SDS/proteinase K-phenol/chloroform extraction.

[0098] 3. Analysis of LOH

[0099] LOH was detected by using VNTR (variable number of tandem repeat)and RFLP probes in the Southern blotting analysis. 10 ug HCC andnoncancerous tissue DNA were digested with proper restriction enzymes,run electrophoresis at 25V for 13 hr, and transferred onto the hybond-Nmembrane (Amersham). YNZ22 probe was given as a gift by Huyin fromShanghai Medical University. Probes 144D6 and YNH37.3 were bought fromAmerican Type Culture Collection (ATCC). Probes cCI17-708 and cCI17-680were given by Dr. Nakamura. The probes were labeled with ((-³²P (dATP ina random priming method. For these probes, the site names and therestriction enzymes were listed in Table 1. Probes YNZ22.1, 144D6 andYNH37.3 were incubated overnight at 42° C. in a conventionalhybridization solution containing 50% formamide. For the hybridizationof Probes cCI17-708 and cCI17-680, pre-hybridization solution was 50%formamide, 5×SSC, 0.5% SDS, 10× Denhardt's solution, 250 ug/ml humanplacent DNA and prehybridization was 42° C. for 24 hr. For hybridizationsolution, except for the replacement of 10× Denhardt's solution by 10%dextran sulfate solution, the other components were identical to thoseof pre-hybridization solution. The hybridization was 42° C. overnight.

[0100] The analysis of microsatellite markers was in a 12.5 ul reactionvolume containing 1 uM PCR primers, 25 ng tissue DNA, 200 uM dNTP, 50 mMKCI, 10 mM Tris (pH9.0), 1.5 mM MgCl₂ 0.625U Taq DNA polymerase(Promega). The names of microsatellite markers and annealingtemperatures were listed in Table 1. The protocol of PCR was 97° C. for4 min; 94° C. for 1 min, annealling at proper temperature for 1 min, and72° C. for 1 min, for 30 cycles; and extending at 72° C. for 10 min. Theproducts were detected in 2% agarose gel electrophoresis. One primer waslabeled with [r-³²P]ATP. In the 5 ul PCR reaction volume, the systemcontained 1 pmol labeled primer, 1 ul above PCR product, 0.25U Taq DNApolymerase. Except that the PCR reaction was carried out 4 cycles, theother reaction conditions and components were the same as above. Afterdenaturation, PCR products were isolated on a 6% denaturingpolyacrylamide gel, and observed for LOH results.

[0101] The LOH for a total of 16 polymorphic markers was detected inchromosome 17p13.3. The LOH frequencies for these markers were listed inTable 1. For each sample, the LOH of each site were summarized in Table2. First, 22 samples were detected for LOH at YNZ22 site and 12 of 19heterozygotes had LOH with a frequency of 63%. Then, 21 samples weredetected for LOH at D17S34 site near telomere and 8 of 12 heterozygoteshad LOH with a frequency 67%. It was of great interest that all of LOHpositive samples at YNZ22 site had LOH at D17S34 site except thehomozygotes, suggesting in HCC samples, the LOH region in 17p13.3 was atleast from YNZ22 site to D17S34 site which was near telomere. Based onthis consideration, 14 markers around these two sites were selected forfurther detection of LOH in HCC samples. The results showed that the 8markers between D17S34 and D17S5 had high LOH in HCC samples, and the 3markers at D17S5 and proximal to centromere had low or no LOH. Thisconfirmed our presumption that the loss region in chromosome 17p13.3 wasfrom D17S34 to D17S5 site in HCC samples. 22 samples were detected usingD17S926 marker. Each of 10 heterozygotes had LOH and the frequency was100%. The samples having LOH at YNZ22 site also had LOH at this siteexcept the homozygotes. D17S1866, D17S849, D17S643, D17S1840, D17S654and D17S1574 markers also were shown LOH in the samples having LOH atYNZ22 site, except the homozygotes and the frequency was 68-86%. ProbeYNH37.3 (D17S28) was used to detect 9 samples having LOH at YNZ22 site.When cleavaged with Taq I, all these 9 samples were homozygotes so thatthe LOH at this site could not be determined. It should be noticed that,in HCC samples NO.16 and NO.34, there was no LOH at YNZ22 site andD17S1574 site proximasl to centromere. In HCC sample NO.16, there was noLOH at D17S34 and D17S1866 sites proximal to centromere. In HCC sampleNO.34, there was no LOH at D17S34, D17S1866 and D17S849 sites proximalto centromere. However, in these two HCC samples, all of the sitesbetween D17S849 and D17S1574 had LOH homozygotes. Therefore, the minimumfrequent loss region in chromosome 17p13.3 in HCC samples was fromD17S849 to D17S1574.

[0102] While analyzing the LOH at various sites in chromosome 17p13.3,the LOH of TP53 site in p53 was also analyzed for chromosome 17p13.1.The results showed that said site had only 31% LOH in HCC, much lowerthan the LOH frequencies of the sites between D17S34 to D17S5 inchromosome 17p13.3 (Tables 1 and 2). TABLE 1 Probe and LOH RESTRIC- PCRNUMBER OF LOH/ TION ANNEALING DETECTED HCC HETEROZYGOTE PROBE SITE NAMEENZYME TEMP. SAMPLES NUMBER % 144D6 D17S34 Rsa I 21  8/12 67 D17S1866*55° C. 22 13/19 68 D17S849* 55° C. 22  9/11 82 D17S926* 58° C. 22 10/10100 D17S695* 60° C. 21 14/18 78 D17S1840* 55° C. 22 6/7 86 D17S1529* 60°C. 33 14/17 82 D17S643* 60° C. 22 12/16 75 D17S831* 60° C. 31 18/24 75D17S654* 65° C. 22 10/13 77 D17S1574* 57° C. 21  9/13 69 YNH37.3 D17S28TaqI 9 0/0 0 YNZ22 D17S5 BamH I 22 12/19 63 D17S525* 59° C. 12 1/4 25cCI17-680 D17S1587 Taq I 6 0/6 0 cCI17-708 D17S878 Msp I 6 0/6 0 TP53*60° C. 22  4/13 31

[0103] TABLE 2 LOH Analysis of chromosome 17P13.3

loss of heterozygosity ◯retention of both alleles

loss of homozygosity

space not analyzed

EXAMPLE 2 Cloning of c63R cDNA

[0104] 1. Screening Genomic PAC Clone for D17s926 in Chromosome 17p13.3:

[0105] The sequences of the D17s926 primers were obtained from Genbank.926-1: GCA GTG GGC CAT CAT CA (SEQ ID NO:3) 926-2: CCG CAG AAG GCT GTTGT (SEQ ID NO:4)

[0106] The primers were sent to Genome systems Inc for screening PAChuman genomic library. Positive clone P579 was obtained.

[0107] 2. A PAC single bacterium was picked from the plate containingP579 (provided by Genome System), inoculated in 5 ml LB containing 25ug/ml kanamycin and shaken overnight at 37° C., 3000 rpm. 2.5 mlovernight culture was added into 75 ml LB, shaken for 1.5 hr. IPTG wasadded to final concentration of 0.5 mM. After cultured under inductioncondition for 5 hr, the bacteria were harvested and centrifuged at10,000 g for 10 min. The pellet of bacteria was used to prepare PAC DNAaccording to the method of extraction of plasmid DNA in “MolecularCloning” (Sambrook, et al., E. F. F. ritsch, T. Maniatis., Translated byJin Dongyan, Li Menfen. 1992, 2nd edition, Science publication). About10 ug p579 DNA was obtained.

[0108] P579 DNA was digested with Not I at 37° C. for 2-3 hr. Theinserts was about 100 Kb. The DNA fragments was recovered from lowmelting-point gel and used as probes.

[0109] 200-300 ng of the above purified DNA fragments were labeled with³²P-α dATP or ³²P-α dCTP in a random priming method according to thespecification of the kit (Megaprime DNA labeling system from Amersham).

[0110] The bacteriophage solution of cDNA library was plated onto 30 150mm—diameter Petri dish at 50,000 pfu/plate, and cultured at 41° C. for3-5 hr. When the diameter of plaques was 0.5 mm and the neighboringplaques were not combined, the Petri dishes were taken out fromincubator, placed at 4° C. for at least 1 hr. The plates were taken outfrom refrigerator and placed in ambient temperature. The NC membrane(Hybond-C Amersham LIFE Science) was gently placed onto the plate. Theinjector filled with ink was used to mark 4 points for orientation. Themembrane was taken, treated by alkaline denaturation for 5 min (0.5 molNaOH—1.5 mol NaCl), neutralized for 5 min (1 mol Tris-HCl pH7.4, 1.5 molNaCl), transferred to 6×SSC, soaked for a while, placed onto 3 mmfiltration paper to dry and baked at 80° C. for 2 hr.

[0111] The membrane was hybridized in pre-hybridization solution at65-68° C. for 6-8 hr. The pre-hybridization solution contained 6×SSC, 5×Denhart's, 0.5% SDS, denatured fish sperm DNA 200 ug/ml, denaturedplacent DNA 100 ug/ml and mixed DNA (γ DNA 5.8, γ DNA 7.3 and PBRplasmid DNA, 30 ug/ml each).

[0112] Into 300 ul solution of labeled probe was added 1 mg Cot-1 DNAand mixed DNA (50 ug each) and 250 ul 20×SSC. The mixture was boiled at100° C. for 15 min, placed onto ice for 10 min, blocking-hybridized in60-65° C. water bath for 20 min, and placed onto ice for 20 min. It wasadded into the pre-hybridization solution containing the membrane andhybridized at 65-68° C. for 16-24 hr. After completion of hybridization,the hybridization solution was discarded and the membrane was rinsedwith 42° C. 2×SSC—0.1% SDS solution twice, each for 10 min. During theprocess of rinse, isotope detector was used for detection so as toadjust the time and temperature for rinsing membrane. After rinse,autoradiography was taken. The same method was used for the 2nd and 3rdround of screening and finally the positive single clone was obtained.The single positive phage was placed into 1 ml SM buffer and a drop ofchloroform was added. It was stored at ambient temperature or 4° C. asphage stock of positive single clone. The positive clone of c63R wasobtained by said screening.

EXAMPLE 3 Construction of c63R Recombinant Plasmid

[0113] 100 ul phage stock and bacteria Y1090 cultured overnight andtreated with 10 mM MgSO₄ were incubated together at 37° C. for 20 min.After addition of 3.5 ml of 0.7% LB agarose (50° C.), the mixture wasplated to plate (90 mm diameter) containing 1% agar medium and incubatedovernight at 37° C. The plate was taken out, and 3 ml SM solution wasadded for each plate. After incubation overnight at 4° C., the solutioncontaining phage was collected.

[0114] Into 150 ul of said collected phage, 1 ml of strain Y1090 treatedwith 10 mM MgSO₄ was added and incubated at 37° C. for 15 min. 40 ml LBcontaining {fraction (1/100)} 0.5M CaCl₂ was added and vigorously shakenat 37° C. until liquid turned from cloudy to clean (about 3 hr). 1 ml ofchloroform was added and at shaken at 37° C. for 20 min, centrifuged at4000 rpm for 10 min. Into supernant was added 50 ul of RNase A/Dnasesolution, and incubated at 37° C. for 1 hr. After overnight incubationat 4° C., it was centrifuged at 1000 rpm for 10 min and the supernantwas super-centrifuged at 27000 rpm for 70 min. Into the pellets wasadded 0.5 ml of SM solution and transferred into 1.5 ml centrifuge tube.5 ul of proteinase K and ⅕ (v/v) 0.5M STE were added. The mixture wasincubated at 50° C. for 30 min, extracted with phenol/chloroform, andprecipitated with alcohol to obtain phage DNA. The DNA was digest withEcoR I, and DNA fragments were recovered using gel recovering method(The Gel Recovering Kit was bought from Huasuo Biotechnology Co.,Shanghai). The DNA of expression vector PBK-CMV (Stratagene) wasdigested with same EcoR I and treated with CIAP enzyme. The DNA insertsand vector PBK-CMV DNA in a molar ratio of 3:1 were ligated with T4 DNAligase at 12° C. overnight, and used to transform competenet cellX_(L)I-blue. The white positive clones were picked out for preparationof plasmid DNA. The enzymatic digestion analysis confirmed the subcloneswere correct.

EXAMPLE 4 Sequencing and Analysis of c63R Nucleic Acid Sequence

[0115] The sequencing of the terminal sequence of c63R clones wascarried out on ABI 337 DNA auto-sequencer using dideoxy chaintermination method. Based on the sequenced sequence, the primers weredesigned and the sequencing was repeated until the full-length sequenceof 2132 bp was obtained. Further analysis showed that the 54 bp at 5′end were from vector. Therefore, the full-length sequence was 2078 bpshown in SEQ ID NO: 1. The ORF was from position 56 to 2077. Thefull-length c63R protein had 673 amino acid as shown in SEQ ID NO: 2.The combination of nucleic acid and amino acid sequence was shown in SEQID NO: 1.

EXAMPLE 5 Gene Cloning from Human cDNA Using PCR

[0116] The human liver tissue was taken, and the total RNA was extractedusing Trizol agents (GIBCO.BRL) according to the specification, and mRNAwas extracted using mRNA Purification Kit (Pharmacia). The reversetranscription was carried out at 42 degree using MMLV-RT-Superscript II(GIBCO BRL) to obtain cDNA. The following ORF specific primers weresynthesized: c63R-1: atgatggaggaggaggaa (SEQ ID NO: 5); c63R-2:gttgtcacggatgatggg (SEQ ID NO: 6). The protocol was 90° C., 3 min, 1cycle; 94° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 35 cycles; 72° C. 10min, 1 cycles. The amplification product containing complete ORFsequence was obtained. The sequencing confirmed the sequence of theproducts was in accord with sequence of Example 4.

EXAMPLE 6 Expression of c63R Gene in Various Tissues

[0117] The c63R fragment was digested and isolated from c63R recombinantplasmid and labeled with α ³²P-dATP using Kit of Megaprime DNA LabelingSystem (Amersham) according to the specification. The labeled probeswere hybridized with multi-tissue mRNA membrane (Biochain). The resultsshowed that there were 3 hybridization bands in various tissues.

EXAMPLE 7 Alteration of C63R Gene in HCC Tissue

[0118] The C63R gene was obtained from chromosome 17p13.3 to furtherdetect the alteration in HCC tissue. The total RNA was extracted fromHCC tissues and noncancerous tissues and turned into cDNA byreverse-transcription.

[0119] The following C63R specific primers were synthesized: C63R-a-PF:CGGGTGGCGGAATGATG (SEQ ID NO:7) C63R-a-PR: CTCCACCCCCATCTACCA (SEQ IDNO:8) C63R-b-PF: GGTGGCGGAATGATGGA (SEQ ID NO:9) C63R-b-PR:CAAAACGCTTCTCCGGC (SEQ ID NO:10) C63R-c-PF: TGCATGGCTGAGAGGATTG (SEQ IDNO:11) C63R-c-PR: ACAACCCCGTGGCTTCC (SEQ ID NO:12) C63R-d-PF:TGCGTACCAGAGCGAAGG (SEQ ID NO:13) C63R-d-PR: CAAGCAGAACGTCCCCAT (SEQ IDNO:14) C63R-a-SR: AGGATGGACGGCAAGCG (SEQ ID NO:15) C63R-b-SR:GGAGGACCCAGCAATGTT (SEQ ID NO:16) C63R-c-SR: CCAAGACAAGAACCTCGGA (SEQ IDNO:17) C63R-d-SR: AACATCCTGAGCACGGCA (SEQ ID NO:18)

[0120] The first PCR was carried out using C63R-a-PF and C63R-a-PR,C63R-c-PF and C63R-c-PR as primers primer and cDNA as template accordingto the following protocol: 94° C., 3 min, 1 cycle; 94° C., 30 sec(denaturation), 60° C. 30 sec (annealing), 72° C., 1 min (extension), 35cycles; 72° C., 10 min (extension). The second nested PCR was carriedout using C63R-b-PF and C63R-b-PR, C63R-d-PF and C63R-d-PR as primersaccording to the above protocol, thereby obtaining the PCR products.

[0121] The PCR products amplified with primers C63R-b-PF and C63R-b-PRwere sequenced with C63R-b-PF, C63R-a-SR, C63R-b-SR, and C63R-b-PR. ThePCR products amplified with primers C63R-d-PF and C63R-d-PR weresequenced with C63R-d-PF, C63R-c-SR, C63R-d-SR and C63R-d-PR.

[0122] The analysis results showed that in HCC Samples 3, 12, 25, 28, 33and G11, there were deletion at nt341-427 but there was no correspondingdeletion in noncancerous tissue. There was deletion of nt1274-1457 inHCC Sample 28 but there was no corresponding deletion in noncanceroustissue. There was deletion of nt1090-1177 in HCC Sample 31 but there wasno corresponding deletion in noncancerous tissue. There was deletion ofnt1271-1365 in HCC Sample x31 but there was no corresponding deletion innoncancerous tissue. There was a mutation from C to T at nt1052 in HCCSample G11, making the amino acid change from Arg to Cys. In HCC SampleG4, there was a deletion of T at nt994, causing frameshift mutation. InHCC Sample GT7, nt743-887 and nt1221-1529 were deleted, causingframeshift mutation. In HCC Sample GT12, nt1612-1769 was deleted,causing frameshift mutation.

[0123] These results indicated that c63R gene was a HCC associated gene.The mutations of c63R gene such as deletion, insertion, and substitutionwere related to HCC. These mutations made c63R protein have no or lowactivity in hepatocyte, finally directly or indirectly causing HCC.

EXAMPLE 8 Genomic Structure and Subcellular Location of HCCS1

[0124] The exon/intron structure of HCCS1 gene was defined by comparingthe cDNA sequences with its genomic sequences. HCCS1 had 18 exons,spanning a genomic sequence of about 200 kb (FIG. 1).

[0125] To identify the subcellular localization of the HCCS1 proteinproduct, the HCCS1-GFP fused gene was cloned into pEGFP-N1 vector andthe HCCS1-GFP plasmid DNA was transfected into both human HCC cell lineSMMC-7721 and mouse NIH/3T3 fibroblasts. The fluorescence image revealedthe punctate distribution of the HCCS1 gene product in the cytoplasm ofboth types of cells. Overlay of the fluorescence image of GFP and anantimitochondria monoclonal antibody, 113-1 (NeoMarkers, Fremont,Calif.), strongly suggested that HCCS1 fusion protein was located inmitochondria (FIG. 2).

EXAMPLE 9 Immunohistochemical Assays

[0126] HCC and noncancerous tissues were fixed in 10% formalin in 10 mMPBS (pH 7.2). The paraffin sections (4 um) were mounted ontopoly-L-lysine-coated glass slides and dried overnight at 50° C. Mouseanti-HCCS1 polyclonal antibody was diluted 1:200 in PBS containing 5%normal goat serum and incubated for 30 min at room temperature. Afterbeing rinsed in PBS three times for 5 min each, the sections werecovered with DAKO EnVision™ +System, horseradish peroxidase(3,3′-diaminobenzidine), Mouse Ready-to-use Detection System (DAKO,Carpinteria, Calif.) for 30 min at room temperature. The sections weredeveloped in substrate-chromogen solution (3,3′-diaminobenzidine),counterstained with Mayer's hematoxylin, and mounted.

[0127] The results were shown in FIG. 3. The HCC was negative and HC,positive.

EXAMPLE 10 Effect on Cell Growth by HCCS1

[0128] According to the manufacturer's protocols, HCCS1 cDNA wasinserted into pcDNA3.1/V5-His vector (Invitrogen), and transfected itinto SMMC-7721 human hepatic carcinoma cells (3×10⁴) by usingLipofectAMINE in triplicate. Empty vector was used as a control. AfterG418 (800 mg/ml) selection for 14 days, the colonies were stained andcounted.

[0129] The results were shown in FIG. 4, indicating HCCS1 inhibited thegrowth of tumor cells.

EXAMPLE 11 Effect on Formation of Tumor in Nude Mice by HCCS1

[0130] Cells harboring HCCS1 cDNA or empty vector were collected andresuspended in PBS. Cells (200 ul; 3.7×10⁶) were inoculated s.c. intothe right flank of 5- to 6-week-old male BALB/c nude mice. Experimentaland control groups had six mice each. After 5 weeks, mice weresacrificed, and the tumors were dissected and weighed.

[0131] The results were shown in FIG. 5, indicating HCCS1 inhibited theformation of tumor in nude mice.

[0132] All the documents cited herein are incorporated into theinvention as reference, as if each of them is individually incorporated.Further, it would be appreciated that, in the above teaching of theinvention, the skilled in the art could make certain changes ormodifications to the invention, and these equivalents would still bewithin the scope of the invention defined by the appended claims of thepresent application.

1 18 1 2078 DNA Homo sapiens CDS (56)..(2077) 1 cggggaggcc tgagttgggctcgcggcggg ggtcggcagg gggccgggtg gcgga atg 58 Met 1 atg gag gag gag gaactg gag ttc gtg gag gag ctg gaa gcc gtg ctg 106 Met Glu Glu Glu Glu LeuGlu Phe Val Glu Glu Leu Glu Ala Val Leu 5 10 15 cag ctc acg ccc gag gtgcag ctg gcc atc gag cag gtg ttt cca agc 154 Gln Leu Thr Pro Glu Val GlnLeu Ala Ile Glu Gln Val Phe Pro Ser 20 25 30 cag gac cct cta gat cga gcagat ttc aat gct gtt gag tat atc aat 202 Gln Asp Pro Leu Asp Arg Ala AspPhe Asn Ala Val Glu Tyr Ile Asn 35 40 45 acc ctg ttc cca acc gag caa tctctg gcg aac ata gac gaa gtc gtg 250 Thr Leu Phe Pro Thr Glu Gln Ser LeuAla Asn Ile Asp Glu Val Val 50 55 60 65 aac aaa att agg ctg aaa ata aggaga ctg gat gac aat att cga act 298 Asn Lys Ile Arg Leu Lys Ile Arg ArgLeu Asp Asp Asn Ile Arg Thr 70 75 80 gtt gta aga ggt cag acg aac gtg gggcag gat gga cgg caa gcg ctt 346 Val Val Arg Gly Gln Thr Asn Val Gly GlnAsp Gly Arg Gln Ala Leu 85 90 95 gaa gag gct cag aaa gct atc caa caa ctcttt ggc aaa atc aaa gat 394 Glu Glu Ala Gln Lys Ala Ile Gln Gln Leu PheGly Lys Ile Lys Asp 100 105 110 atc aaa gac aaa gct gaa aaa tca gag caaatg gtg aaa gaa atc acc 442 Ile Lys Asp Lys Ala Glu Lys Ser Glu Gln MetVal Lys Glu Ile Thr 115 120 125 cgt gat att aag caa tta gat cac gcc aaacgc cac ctg acc acc tca 490 Arg Asp Ile Lys Gln Leu Asp His Ala Lys ArgHis Leu Thr Thr Ser 130 135 140 145 atc acc aca ctg aac cac ctg cac atgctg gca gga ggt gtc gac tcc 538 Ile Thr Thr Leu Asn His Leu His Met LeuAla Gly Gly Val Asp Ser 150 155 160 ctc gaa gcc atg acc agg cga aga caatac gga gaa gtt gct aat ctc 586 Leu Glu Ala Met Thr Arg Arg Arg Gln TyrGly Glu Val Ala Asn Leu 165 170 175 ctt cag ggt gtg atg aat gtc ctg gagcac ttc cac aag tat atg ggg 634 Leu Gln Gly Val Met Asn Val Leu Glu HisPhe His Lys Tyr Met Gly 180 185 190 att ccg cag atc cgg cag ctt tcc gaaaga gtg aag gct gca cag act 682 Ile Pro Gln Ile Arg Gln Leu Ser Glu ArgVal Lys Ala Ala Gln Thr 195 200 205 gag tta gga cag caa atc ctg gca gatttt gaa gaa gcg ttt cct tcc 730 Glu Leu Gly Gln Gln Ile Leu Ala Asp PheGlu Glu Ala Phe Pro Ser 210 215 220 225 cag ggc acc aag aga cca gga ggaccc agc aat gtt cta cga gat gca 778 Gln Gly Thr Lys Arg Pro Gly Gly ProSer Asn Val Leu Arg Asp Ala 230 235 240 tgt ctg gtt gct aat att cta gatccc agg atc aaa cag gaa atc atc 826 Cys Leu Val Ala Asn Ile Leu Asp ProArg Ile Lys Gln Glu Ile Ile 245 250 255 aaa aag ttt att aaa cag cat ctgtca gag tat ctg gta ctt ttt caa 874 Lys Lys Phe Ile Lys Gln His Leu SerGlu Tyr Leu Val Leu Phe Gln 260 265 270 gaa aac caa gat gtt gcc tgg ctggac aaa atc gac aga cgc tat gcc 922 Glu Asn Gln Asp Val Ala Trp Leu AspLys Ile Asp Arg Arg Tyr Ala 275 280 285 tgg ata aaa cgc cag ctt gtg gactat gag gag aaa tac ggc cgc atg 970 Trp Ile Lys Arg Gln Leu Val Asp TyrGlu Glu Lys Tyr Gly Arg Met 290 295 300 305 ttt cca cgt gag tgg tgc atggct gag agg att gcg gtg gaa ttt tgc 1018 Phe Pro Arg Glu Trp Cys Met AlaGlu Arg Ile Ala Val Glu Phe Cys 310 315 320 cat gtg aca agg gca gaa cttgcc aag att atg cgt acc aga gcg aag 1066 His Val Thr Arg Ala Glu Leu AlaLys Ile Met Arg Thr Arg Ala Lys 325 330 335 gaa att gaa gtg aaa ttg cttctt ttt gct att caa aga aca act aac 1114 Glu Ile Glu Val Lys Leu Leu LeuPhe Ala Ile Gln Arg Thr Thr Asn 340 345 350 ttt gag ggg ttt ctt gca aaacgc ttc tcc ggc tgc acc ctg acc gat 1162 Phe Glu Gly Phe Leu Ala Lys ArgPhe Ser Gly Cys Thr Leu Thr Asp 355 360 365 ggg acc ctg aaa aag ctt gagtct cca ccc cca tct acc aat ccc ttc 1210 Gly Thr Leu Lys Lys Leu Glu SerPro Pro Pro Ser Thr Asn Pro Phe 370 375 380 385 ctg gaa gat gag cca acacca gag atg gag gaa ctg gca acg gag aaa 1258 Leu Glu Asp Glu Pro Thr ProGlu Met Glu Glu Leu Ala Thr Glu Lys 390 395 400 gga gat tta gat caa ccaaag aag cct aaa gcc cca gac aat cca ttt 1306 Gly Asp Leu Asp Gln Pro LysLys Pro Lys Ala Pro Asp Asn Pro Phe 405 410 415 cat ggc att gtt tcc aagtgt ttt gag cct cat ctc tac gtg tat atc 1354 His Gly Ile Val Ser Lys CysPhe Glu Pro His Leu Tyr Val Tyr Ile 420 425 430 gaa tcc caa gac aag aacctc gga gag ctg ata gat cgg ttt gtg gct 1402 Glu Ser Gln Asp Lys Asn LeuGly Glu Leu Ile Asp Arg Phe Val Ala 435 440 445 gat ttc aaa gcc cag gggcca cct aag ccc aac act gat gaa ggg ggt 1450 Asp Phe Lys Ala Gln Gly ProPro Lys Pro Asn Thr Asp Glu Gly Gly 450 455 460 465 acc gtg ctc ccc agctgc gcc gac ctc ttt gtc tac tac aag aag tgc 1498 Thr Val Leu Pro Ser CysAla Asp Leu Phe Val Tyr Tyr Lys Lys Cys 470 475 480 atg gtg caa tgc tctcag ctc agt act ggg gag ccc atg atc gcc ctg 1546 Met Val Gln Cys Ser GlnLeu Ser Thr Gly Glu Pro Met Ile Ala Leu 485 490 495 acc acc att ttc cagaag tac ctc cga gaa tac gcc tgg aaa atc ctc 1594 Thr Thr Ile Phe Gln LysTyr Leu Arg Glu Tyr Ala Trp Lys Ile Leu 500 505 510 tct ggc aac ctg cccaaa ccc aca acc agc agt gaa gga ctg act atc 1642 Ser Gly Asn Leu Pro LysPro Thr Thr Ser Ser Glu Gly Leu Thr Ile 515 520 525 agc agc ctc ctc aaggaa aag gag ggc tca gaa gta gcc aag ttc act 1690 Ser Ser Leu Leu Lys GluLys Glu Gly Ser Glu Val Ala Lys Phe Thr 530 535 540 545 ctg gag gag ctctgc ctc atc tgt aac atc ctg agc acg gca gag tac 1738 Leu Glu Glu Leu CysLeu Ile Cys Asn Ile Leu Ser Thr Ala Glu Tyr 550 555 560 tgt ctg gcc accacc cag cag cta gaa gaa aaa ctc aaa gaa aaa gtg 1786 Cys Leu Ala Thr ThrGln Gln Leu Glu Glu Lys Leu Lys Glu Lys Val 565 570 575 gat gta agt ctgatt gaa cga atc aat ctg act gga gag atg gac acg 1834 Asp Val Ser Leu IleGlu Arg Ile Asn Leu Thr Gly Glu Met Asp Thr 580 585 590 ttc agc acc gtcatc tcc agc agt att cag ctg ctg gtt cag gat ctg 1882 Phe Ser Thr Val IleSer Ser Ser Ile Gln Leu Leu Val Gln Asp Leu 595 600 605 gat gct gcc tgtgat cct gcc ctg act gcc atg agc aag atg cag tgg 1930 Asp Ala Ala Cys AspPro Ala Leu Thr Ala Met Ser Lys Met Gln Trp 610 615 620 625 cag aac gtggag cac gtt ggt gac cag agc ccc tac gtc acc tct gtc 1978 Gln Asn Val GluHis Val Gly Asp Gln Ser Pro Tyr Val Thr Ser Val 630 635 640 att ctg cacatc aag cag aac gtc ccc atc atc cgt gac aac ccc gtg 2026 Ile Leu His IleLys Gln Asn Val Pro Ile Ile Arg Asp Asn Pro Val 645 650 655 gct tcc acacgc aag tac ttc act cag tta tgc gtt aaa ttt gca aag 2074 Ala Ser Thr ArgLys Tyr Phe Thr Gln Leu Cys Val Lys Phe Ala Lys 660 665 670 taa a 2078 2673 PRT Homo sapiens 2 Met Met Glu Glu Glu Glu Leu Glu Phe Val Glu GluLeu Glu Ala Val 1 5 10 15 Leu Gln Leu Thr Pro Glu Val Gln Leu Ala IleGlu Gln Val Phe Pro 20 25 30 Ser Gln Asp Pro Leu Asp Arg Ala Asp Phe AsnAla Val Glu Tyr Ile 35 40 45 Asn Thr Leu Phe Pro Thr Glu Gln Ser Leu AlaAsn Ile Asp Glu Val 50 55 60 Val Asn Lys Ile Arg Leu Lys Ile Arg Arg LeuAsp Asp Asn Ile Arg 65 70 75 80 Thr Val Val Arg Gly Gln Thr Asn Val GlyGln Asp Gly Arg Gln Ala 85 90 95 Leu Glu Glu Ala Gln Lys Ala Ile Gln GlnLeu Phe Gly Lys Ile Lys 100 105 110 Asp Ile Lys Asp Lys Ala Glu Lys SerGlu Gln Met Val Lys Glu Ile 115 120 125 Thr Arg Asp Ile Lys Gln Leu AspHis Ala Lys Arg His Leu Thr Thr 130 135 140 Ser Ile Thr Thr Leu Asn HisLeu His Met Leu Ala Gly Gly Val Asp 145 150 155 160 Ser Leu Glu Ala MetThr Arg Arg Arg Gln Tyr Gly Glu Val Ala Asn 165 170 175 Leu Leu Gln GlyVal Met Asn Val Leu Glu His Phe His Lys Tyr Met 180 185 190 Gly Ile ProGln Ile Arg Gln Leu Ser Glu Arg Val Lys Ala Ala Gln 195 200 205 Thr GluLeu Gly Gln Gln Ile Leu Ala Asp Phe Glu Glu Ala Phe Pro 210 215 220 SerGln Gly Thr Lys Arg Pro Gly Gly Pro Ser Asn Val Leu Arg Asp 225 230 235240 Ala Cys Leu Val Ala Asn Ile Leu Asp Pro Arg Ile Lys Gln Glu Ile 245250 255 Ile Lys Lys Phe Ile Lys Gln His Leu Ser Glu Tyr Leu Val Leu Phe260 265 270 Gln Glu Asn Gln Asp Val Ala Trp Leu Asp Lys Ile Asp Arg ArgTyr 275 280 285 Ala Trp Ile Lys Arg Gln Leu Val Asp Tyr Glu Glu Lys TyrGly Arg 290 295 300 Met Phe Pro Arg Glu Trp Cys Met Ala Glu Arg Ile AlaVal Glu Phe 305 310 315 320 Cys His Val Thr Arg Ala Glu Leu Ala Lys IleMet Arg Thr Arg Ala 325 330 335 Lys Glu Ile Glu Val Lys Leu Leu Leu PheAla Ile Gln Arg Thr Thr 340 345 350 Asn Phe Glu Gly Phe Leu Ala Lys ArgPhe Ser Gly Cys Thr Leu Thr 355 360 365 Asp Gly Thr Leu Lys Lys Leu GluSer Pro Pro Pro Ser Thr Asn Pro 370 375 380 Phe Leu Glu Asp Glu Pro ThrPro Glu Met Glu Glu Leu Ala Thr Glu 385 390 395 400 Lys Gly Asp Leu AspGln Pro Lys Lys Pro Lys Ala Pro Asp Asn Pro 405 410 415 Phe His Gly IleVal Ser Lys Cys Phe Glu Pro His Leu Tyr Val Tyr 420 425 430 Ile Glu SerGln Asp Lys Asn Leu Gly Glu Leu Ile Asp Arg Phe Val 435 440 445 Ala AspPhe Lys Ala Gln Gly Pro Pro Lys Pro Asn Thr Asp Glu Gly 450 455 460 GlyThr Val Leu Pro Ser Cys Ala Asp Leu Phe Val Tyr Tyr Lys Lys 465 470 475480 Cys Met Val Gln Cys Ser Gln Leu Ser Thr Gly Glu Pro Met Ile Ala 485490 495 Leu Thr Thr Ile Phe Gln Lys Tyr Leu Arg Glu Tyr Ala Trp Lys Ile500 505 510 Leu Ser Gly Asn Leu Pro Lys Pro Thr Thr Ser Ser Glu Gly LeuThr 515 520 525 Ile Ser Ser Leu Leu Lys Glu Lys Glu Gly Ser Glu Val AlaLys Phe 530 535 540 Thr Leu Glu Glu Leu Cys Leu Ile Cys Asn Ile Leu SerThr Ala Glu 545 550 555 560 Tyr Cys Leu Ala Thr Thr Gln Gln Leu Glu GluLys Leu Lys Glu Lys 565 570 575 Val Asp Val Ser Leu Ile Glu Arg Ile AsnLeu Thr Gly Glu Met Asp 580 585 590 Thr Phe Ser Thr Val Ile Ser Ser SerIle Gln Leu Leu Val Gln Asp 595 600 605 Leu Asp Ala Ala Cys Asp Pro AlaLeu Thr Ala Met Ser Lys Met Gln 610 615 620 Trp Gln Asn Val Glu His ValGly Asp Gln Ser Pro Tyr Val Thr Ser 625 630 635 640 Val Ile Leu His IleLys Gln Asn Val Pro Ile Ile Arg Asp Asn Pro 645 650 655 Val Ala Ser ThrArg Lys Tyr Phe Thr Gln Leu Cys Val Lys Phe Ala 660 665 670 Lys 3 17 DNASynthetic primer 3 gcagtgggcc atcatca 17 4 17 DNA Synthetic primer 4ccgcagaagg ctgttgt 17 5 18 DNA Synthetic primer 5 atgatggagg aggaggaa 186 18 DNA Synthetic primer 6 gttgtcacgg atgatggg 18 7 17 DNA Syntheticprimer 7 cgggtggcgg aatgatg 17 8 18 DNA Synthetic primer 8 ctccacccccatctacca 18 9 17 DNA Synthetic primer 9 ggtggcggaa tgatgga 17 10 17 DNASynthetic primer 10 caaaacgctt ctccggc 17 11 19 DNA Synthetic primer 11tgcatggctg agaggattg 19 12 17 DNA Synthetic primer 12 acaaccccgt ggcttcc17 13 18 DNA Synthetic primer 13 tgcgtaccag agcgaagg 18 14 18 DNASynthetic primer 14 caagcagaac gtccccat 18 15 17 DNA Synthetic primer 15aggatggacg gcaagcg 17 16 18 DNA Synthetic primer 16 ggaggaccca gcaatgtt18 17 19 DNA Synthetic primer 17 ccaagacaag aacctcgga 19 18 18 DNASynthetic primer 18 aacatcctga gcacggca 18

What is claimed is:
 1. An isolated human c63R polypeptide comprising apolypeptide having the amino acid sequence of SEQ ID NO: 2, itsconservative variants, its active fragments, and its active derivatives.2. The polypeptide of claim 1 wherein said polypeptide is a polypeptidehaving the amino acid sequence of SEQ ID NO:
 2. 3. An isolatedpolynucleotide comprising a nucleotide sequence sharing at least 85%homology to a nucleotide sequence selected from the group consisting of:(a) the nucleotide sequence encoding the c63R polypeptide of claims 1 or2; (b) the polynucleotide complementary to nucleotide sequence of (a).4. The polynucleotide of claim 3 wherein said nucleotide sequenceencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
 5. The polynucleotide of claim 3 wherein said nucleotide sequencecomprises the ORF sequence or full-length sequence of SEQ ID NO:
 1. 6. Avector containing the DNA sequence of claim
 3. 7. A geneticallyengineered host cell which is selected from the group consisting of: (a)a host cell transformed with the vector of claim 6, and (b) a host celltransformed with the polynucleotide of claim
 3. 8. A method forproducing a polypeptide having the activity of c63R protein, whichcomprises: (a) culturing the host cell of claim 7 under the conditionssuitable for the expression of protein; (b) isolating the polypeptideshaving the activity of c63R protein from the culture.
 9. An antibodyspecifically bound with the c63R polypeptide of claim
 1. 10 A nucleicacid molecule comprising about 8-100 consecutive nucleotides of thepolynucleotide of claim
 3. 11. A pharmaceutical composition comprising asafe and efficient amount of the polypeptide of claim 1 andpharmaceutically acceptable carrier.
 12. A method for detecting thecarcinomatous change or cancer susceptibility of hepatocytes comprisingthe steps of: detecting whether there is any change of c63R transcriptin the hepatocyte sample when compared with the normal c63R transcript,and said change indicating that the hepatocyte has developed cancer oris cancer susceptible; or detecting whether there is any change ofactivity of c63R protein in the hepatocyte sample when compared with thenormal c63R protein, and said change indicating that the hepatocyte hasdeveloped cancer or is cancer susceptible.
 13. The method of claim 12wherein said change is nucleotide deletion, insertion or substitution.14. The method of claim 13 wherein said change is selected from thegroup consisting of: deletion of nucleotides 341-427 in SEQ ID NO: 1,deletion of nucleotides 1274-1457 in SEQ ID NO: 1, deletion ofnucleotides 1090-1177 in SEQ ID NO: 1, deletion of nucleotides 1271-1365in SEQ ID NO: 1, change from C to T of nucleotide 1052 in SEQ ID NO: 1,deletion of nucleotide 994 in SEQ ID NO: 1, deletion of nucleotides743-887 in SEQ ID NO: 1, deletion of nucleotides 1221-1529 in SEQ ID NO:1, deletion of nucleotide 1612-1769 in SEQ ID NO:
 1. 15. A kit fordetecting hepatocarcinoma comprising the steps of: (1) a pair of primersspecifically amplifying human c63R gene, and (2) the agents fordetecting whether there is any change between the amplification productand the normal c63R gene.